Comparison of ultrasonography-based masticatory muscle thickness between temporomandibular disorders bruxers and temporomandibular disorders non-bruxers

To compare masticatory muscle thickness in patients with temporomandibular disorders (TMDs) during rest and clenching, and by body position, using ultrasonography. This prospective study included 96 patients with TMD (67 females, 29 males; mean age: 40.41 ± 17.88 years): group 1, comprising 66 patients with TMD without bruxism (TMD_nonbruxer), and group 2, comprising 30 patients with concurrent TMD and bruxism (TMD_bruxer). In patients with TMD, bruxism was correlated with the presence of tinnitus, muscle stiffness, sleep problems, psychological stress, and restricted mouth opening. The masseter muscle significantly thickened during clenching (11.16 ± 3.03 mm vs 14.04 ± 3.47 mm, p < 0.001), whereas the temporalis muscle showed no significant increase in thickness from resting to clenching in an upright position (7.91 ± 1.98 vs 8.39 ± 2.08, p = 0.103). Similarly, during clenching in the supine position, the masseter muscle was significantly thicker compared with rest (11.24 ± 2.42 vs 13.49 ± 3.09, p < 0.001), but no significant difference was observed in temporal muscle thickness (8.21 ± 2.16 vs 8.43 ± 1.94, p = 0.464). In comparison between two groups, the average thickness of the masseter muscle was greater among TMD_bruxers than among TMD_nonbruxers in both the upright and supine positions (all p < 0.05). In the generalized lineal model, female sex (B = − 1.018, 95% confidence interval [CI] − 1.855 to − 0.181, p = 0.017) and bruxism (B = 0.868, 95% CI 0.567 to 1.169, p = 0.048) significantly predicted changes in masseter muscle thickness. Female sex (B = − 0.201, 95% CI − 0.299 to − 0.103, p = 0.011), increased age (B = − 0.003, 95% CI − 0.005 to 0.000, p = 0.038), and muscle stiffness (B = − 1.373, 95% CI − 2.369 to − 0.376, p = 0.007) were linked to decreased temporal muscle thickness. Comparing TMD nonbruxer and bruxer muscle thicknesses in upright and supine positions revealed significant increased thickness in the masseter muscle during clenching but not in the temporalis muscle. Masseter muscle thickness varied significantly by sex, body position, and resting/clenching, notably influenced by bruxism. These findings emphasize the relevance of these factors in clinical examinations of patients with TMD.


Participants
Ninety-six patients with TMD (67 females, 29 males; mean age, 40.41 ± 17.88 years) participated in this study at the Department of Orofacial Pain and Oral Medicine of the Kyung Hee University Dental Hospital (Seoul, South Korea) between June 2022 and March 2023.All patients underwent thorough examination and diagnosis by two TMD specialists with > 8 years of experience, using the DC/TMD examination.Additionally, patients completed comprehensive questionnaires, including the DC/TMD symptom questionnaire, DC/TMD demographics, and an oral behavior checklist.For the sample size calculation, we used the G*Power software (latest ver.3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany); 46 subjects (23 subjects per group, actual power = 0.95) were derived with an actual target of at least 30 per group as suitable for statistical analysis, and finally a total of 96 participants were recruited.
Participant selection was based on a standardized clinical examination.The inclusion criteria were as follows: all participants underwent a physical examination according to the DC/TMD 3 and were aged ≥ 18 years.Consequently, individuals diagnosed with arthrogenous and/or myogenous TMD were included in the study.The exclusion criteria were: (1) age < 18 years, (2) history of facial tumor or surgery, (3) local facial infection, (4) systemic inflammatory connective tissue disease, (5) other systemic musculoskeletal disorders (fibromyalgia, rheumatoid arthritis, inflammatory joint disease), (6) neurologic impairment or diseases (stroke, tumor, epilepsy), (7) psychiatric disorders, (8) pregnancy, and (9) inability to provide informed consent.

Clinical evaluation (1) Characteristics of TMD pain
The duration of pain due to TMD has been reported in months.When the symptom duration was > 6 months, it was classified as TMD chronicity 4 .TMD pain was scored by the patients on a visual analog scale (VAS) ranging from 0 (no pain at all) to 10 (worst pain imaginable).The presence of preauricular pain was noted when the patient reported pain in or around the ear along with TMD pain 36 .
(2) Evaluation of bruxism Bruxism is a movement disorder characterized by grinding and/or clenching of teeth, which may occur during wakefulness or sleep.In our assessment, patients were identified as having bruxism if they self-reported bruxism, indicating a positive response to two or more questions, including the initial inquiry.
Questionnaire for detecting bruxer 37 : (1) Has anyone observed or heard your teeth grinding at night? (2) Do you experience jaw fatigue or soreness upon waking up in the morning?
(3) Do you notice any soreness in your teeth or gums upon waking up in the morning?(4) Do you experience temporal headaches upon waking up in the morning?(5) Are you aware of tooth grinding during the day?(6) Do you experience episodes of tooth clenching during the day?
These questions constitute the questionnaire used to identify potential bruxism, aiming to capture various symptoms and manifestations associated with tooth grinding and clenching throughout the day and night.Thus, in this study, bruxism encompasses probable bruxism and refers to both awake bruxism and/or sleep bruxism 12 .
(3) Diagnosis of TMD and TMD subgroups TMD was diagnosed according to DC/TMD 3 .It can be broadly categorized into arthrogenous and myogenous types, depending on the origin and pattern of pain 4 .The three TMD subgroups consisted of arthrogenous TMD, myogenous TMD, and mixed TMD, which presents elements of both.Arthrogenic TMD includes conditions such as arthralgia, arthritis, osteoarthritis, and articular disc displacement.Myogenous TMD comprises local myalgia, myofascial pain, and myofascial pain upon referral.Mixed TMD refer to the coexistence of arthrogenous and myogenous TMD.

(4) Chief complaints of TMD patients
The chief complaints of TMD patients were identified in five categories: TMJ noise, TMD pain, limited mouth opening, muscle stiffness, and occlusal dysesthesia.Chief complaints were organized into one or two or more multiples based on patient reports.TMJ noise was considered present when clicking, fine and/or coarse crepitus, and popping noises in the TMJ occurred during mandibular movement 38 .TMD pain encompassed pain around the TMJ, muscles, ears, and temple areas 39 .Limited mouth opening referred to a situation where comfortable mouth opening was < 35 mm 40 , leading to difficulty or discomfort when opening the mouth.Muscle stiffness in the masticatory muscles was referred to as the sensation of tightness or pain in the muscles 41 .Occlusal dysesthesia described the perpetual perception of disturbing or unpleasant tooth contacts 42 .
(5) Contributing factors or comorbidities for TMD The presence of psychological stress was evaluated using a dichotomous question: "Have you encountered any mental stress or psychological pressure in your daily life over the past week?"Additionally, self-assessed variables including unilateral chewing, preference for hard food, sleep problems, and tinnitus were reported using binary responses (yes/no).All variables were recorded in binary format for all patients, following the methodology detailed in our previous study 6 .(6) Ultrasonography measurements Ultrasonography was performed using an Alpinion E-Cube 8 ultrasound system (Alpinion Medical Systems Co., Ltd., Seoul, Korea) paired with an M12L linear transducer operating at a pulse frequency range of 0-14 MHz.A fixed B-mode setting for grayscale was chosen specifically for musculoskeletal assessments.Individual adjustments for gain, focus, and depth were made for each patient.Doppler sensitivity was optimized for low flow with fixed settings (7.5 MHz Doppler frequency, pulse repetition frequency of 0.9 kHz, wall filter of 114 Hz).Ultrasound images of the major masticatory muscles were captured at specified locations (Fig. 2), allowing for the measurement of muscle thickness and cross-sectional area on both right and left sides.Measurements were performed using a program built into the Alpinion E-Cube 8 ultrasound system.Dental investigators (YHL and YHC) with relevant training conducted all ultrasound examinations.(7) Thickness of the masticatory muscle The masseter and temporalis muscles were initially assessed while the patient was upright and subsequently in a relaxed supine position.To start, the facial area and body were relaxed, the upper and lower lips were gently brought together, and measurements were recorded in both the resting and clenched states.Measurements commenced on the right side of the patient, where the operator was positioned, then proceeded to the left side once the right-side measurements were completed.Both the right and left sides were examined for these two masticatory muscles.Following completion of measurements in the upright posture, the same process was repeated with the patient in the supine position.This protocol aimed to determine any significant differences in muscle thickness between the upright and supine positions, as well as between the resting and clenched states.Regarding the echogenicity of major anatomical structures, normal bone interfaces appeared hyperechoic, fascia was also hyperechoic, and muscles displayed hypoechoic characteristics 43,44 .Measurement of the masseter muscle thickness was conducted at its thickest point beneath the midsection of the lower zygomatic arch.The transducer was positioned over the most prominent part of the masseter muscle, aligned parallel to the long axis of the zygomatic arch, between the lower mandibular notch and inferior border of the mandible.For the temporalis muscle, a linear probe was placed parallel to the outer edge of the eyebrow (Fig. 1).Muscle thickness was defined as the maximum distance between the outer and inner fasciae.The obtained muscle thickness in each condition for one patient represented the average of the measurements from both sides, and the values were recorded in millimeters.
(8) Reliability and measurement errors Inter-and intra-observer reliabilities were used to assess the degree of agreement among multiple repetitions of a clinical test.All ultrasound scan parameters were measured twice by two investigators (YHL and YHC), and images were randomly presented to assess inter-and intra-observer reliability.Inter-class correlation coefficients (ICCs) were calculated, with a predefined threshold set at > 0.80 for the correlations of all items.Intra-examiner reproducibility estimated ICCs of 0.84 and 0.92, respectively.Instances where the ICC did not meet this standard value were earmarked for additional investigation and subjected to re-measurement.Disagreements were resolved through several discussions until a consensus was reached.The ICC values ranged from 0 (no reliability) to 1 (perfect reliability) 45 .Across repeated testing, the ICC consistently met the criterion (> 0.80) in all cases.

Statistical methods
Data were analyzed using SPSS Statistics version 26.0 for Windows (IBM Corp., Armonk, NY).Continuous variables are presented as means and standard deviations (SD), and categorical variables as frequencies and percentages.The intra-rater reliability of muscle thickness and cross-sectional area measurements was assessed using the ICC coefficient, with a mean value of 0.87.Depending on the presence of bruxism in patients, they were categorized into two groups: group 1 (TMD without bruxism, TMD_nonbruxers) and 2 (TMD with bruxism, TMD_bruxers).Chi-square tests examined categorical variable distributions.Bonferroni-adjusted post-hoc analyses were used for chi-square tests to determine the equality of proportions.The thickness of masticatory muscles, including the masseter and temporalis muscles, was measured during both resting and clenching states.Differences in average thickness were investigated based on the presence or absence of bruxism and by sex using a t-test.Spearman's correlation coefficients (r) were calculated for VAS, demographics, and ultrasonographic parameters, ranging from − 1 to + 1, with − 1 representing a perfect linear negative correlation, and + 1 representing a perfect linear positive correlation.A generalized linear model assessed factors affecting thickness changes in the masseter and temporalis muscles.The general linear model is an extension of multiple linear regression for situations involving multiple dependent variables.The extent to which the explanatory variables impacted the outcome variable was represented by the beta coefficient (B) and 95% CI values.A two-tailed p value of < 0.05 was considered statistically significant.

Institutional review board approval
The research protocol adhered to the Declaration of Helsinki and received approval from the Institutional Review Board of Kyung Hee University Dental Hospital in Seoul, South Korea (KHD IRB, IRB No-KH-DT22015).

Informed consent
Informed consent was obtained from all participants.

Distribution of chief complaints and contributing factors for TMD
When examining chief complaints in patients with TMD, differences emerged between the TMD_nonbruxer and TMD_bruxer groups, particularly in limited mouth opening and muscle stiffness.Limited mouth opening (< 35 mm) was significantly more prevalent in the TMD_bruxer group than in the TMD_nonbruxer group (26.7% vs. 9.1%, p = 0.032).Similarly, muscle stiffness was significantly higher in TMD_bruxers than in TMD_nonbruxers (53.3% vs. 24.2%,p = 0.009).However, no significant differences were noted in TMJ noise, TMD pain, and occlusal dysesthesia between the two groups.

Muscle thickness during resting and clenching
This study investigated variations in masseter and temporalis muscle thickness during resting and clenching considering patient body position.Initially, muscle thickness was measured in both the upright and supine positions.In the upright position, the masseter muscle exhibited significantly greater thickness during clenching than during rest (11.16 ± 3.03 vs 14.04 ± 3.47, p < 0.001), marking a difference of 2.92 ± 2.24 mm.Conversely, transitioning from rest to clenching in the upright position did not yield a substantial increase in temporal muscle thickness (7.91 ± 1.98 vs 8.39 ± 2.08, p = 0.103), with a marginal difference of 0.48 ± 1.21 mm.Similarly, in the supine position, the masseter muscle exhibited significantly greater thickness during clenching than during rest (11.24 ± 2.42 vs 13.49 ± 3.09, p < 0.001), showcasing an average thickness difference of 2.25 ± 1.90 mm.However, in the supine position, no significant difference was observed in temporal muscle thickness between resting and clenching states (8.21 ± 2.16 vs 8.43 ± 1.94, p = 0.464) (Table 3).

Muscles thickness of TMD_bruxers in upright position
Comparing muscle thickness between TMD_nonbruxers and TMD_bruxers in both upright and supine positions revealed that only the masseter muscle exhibited a significant increase in thickness during clenching compared with resting (Fig. 3).The study measured the thicknesses of the masseter and temporal muscles, key masticatory muscles, in resting and clenching states for both TMD_non-bruxers and TMD_bruxers in the upright and supine positions.In all cases, the masticatory muscle thickness was greater in TMD bruxers compared with  www.nature.com/scientificreports/TMD non-bruxers, particularly during clenching rather than at rest.However, the differences in muscle thickness between the two conditions were significant only in the masseter muscle, not in the temporalis muscle.A significant disparity in masticatory muscle thickness was observed in the masseter muscle between TMD_ non-bruxers and TMD_bruxers.In the upright position, the masseter muscle was significantly thicker in TMD bruxers than in TMD_nonbruxers during resting (10.44 ± 2.77 mm vs. 12.73 ± 3.03 mm, p < 0.001).Furthermore, during clenching in the upright position, the masseter muscle thickness in TMD bruxers increased significantly compared with TMD non-bruxers (13.48 ± 3.41 mm vs 15.26 ± 3.39 mm, p = 0.021).Regarding the temporal muscles, no significant differences were observed in muscle thickness between TMD_nonbruxers and TMD_bruxers during resting (7.59 ± 1.90 vs 8.05 ± 2.01 mm, p = 0.284) or clenching (7.89 ± 1.96 mm vs 8.61 ± 2.12 mm, p = 0.114) in the upright position (Table 4).www.nature.com/scientificreports/

Muscle thickness of TMD_bruxer in supine position
To discern positional trends, the procedures conducted in the upright position were replicated in the supine position.The observed tendency in the upright position mirrored that observed in the supine position.Even in the supine position, a significant difference in muscle thickness between TMD_nonbruxers and TMD_bruxers was evident solely in the masseter muscles.During supine position at rest, the masseter muscle was significantly thicker in TMD_ bruxers than in TMD_nonbruxers (10.93 ± 2.55 vs. 11.92 ± 1.97 mm, p = 0.043).When clenching in the supine position, the masseter muscle thickness significantly increased in TMD_bruxers compared with TMD_nonbruxers (12.97 ± 3.05 vs 14.62 ± 2.90 mm, p = 0.014).This implies that patients with TMD with bruxism exhibited thicker masseter muscles in the supine position, both at rest and during clenching, compared with those without bruxism.Regarding the temporalis muscles, no statistically significant difference in muscle thickness between TMD_nonbruxers and TMD_bruxers during resting (7.74 ± 1.85 vs 8.43 ± 2.27 mm, p = 0.284) or clenching (8.04 ± 1.86 mm vs 8.61 ± 1.96 mm, p = 0.114) in the supine position.When comparing the muscle thickness of the temporalis muscle between TMD. _nonbruxers and TMD_bruxers in the supine position, no significant difference was found during resting (7.74 ± 1.85 vs 8.43 ± 2.27 mm, p = 0.284) or clenching (8.04 ± 1.86 mm vs 8.61 ± 1.96 mm, p = 0.114).In other words, no discernable difference existed in the thickness of the temporalis muscle between the TMD_nonbruxers and TMD_bruxers either at rest or during clenching in the supine position (Table 4).

Sex-differences in muscle thickness
This study investigated differences in masseter and temporalis muscle thickness between males and females across various body positions, specifically comparing muscle states during rest and clenching.Males generally exhibited greater thickness in both the masseter and temporalis muscles than females, with statistical significance observed primarily in the masseter muscle.When assessing muscle thickness based on sex, in the upright position, the masseter muscle was notably thicker in males than in females only at rest.However, in the supine position, the masseter muscle was significantly thicker in men than in women, both at rest and in the supine position (all p < 0.05).Conversely, no significant difference in thickness between sexes was noted in the temporalis muscle, irrespective of body position and resting/clenching conditions (Fig. 4).
Specifically, in the upright resting position, males exhibited significantly thicker masseter muscles compared with females (12.46 ± 3.14 vs 10.59 ± 2.83 mm, p = 0.008).Interestingly, in the supine position, no significant mean differences were observed in masseter and temporalis muscle thicknesses between sexes.During both resting (12.43 ± 2.74 vs 10.72 ± 2.08, p = 0.004) and clenching (15.23 ± 3.35 vs 12.73 ± 2.66, p < 0.001) in the upright position, males exhibited significantly thicker masseter muscles than females, indicating substantial distinctions.However, no sex-based differences in temporalis muscle thickness were observed during both resting and clenching, irrespective of body position.

Comparison of muscle thickness between TMD subgroups
Based on the DC/TMD classification, patients were categorized into arthrogenous TMD (n = 32), myogenous TMD (n = 31), and mixed TMD (n = 33) groups.Analysis of masseter and temporalis muscle thickness, considering patient posture and resting versus clenching statuses, revealed no statistically significant differences.Across www.nature.com/scientificreports/ the three TMD subgroups (arthrogenous, myogenous, and mixed), both masseter and temporal muscles tended to be thicker during clenching than during the resting state.However, these differences did not reach statistical significance (Table 5).

Correlation coefficient (r) between thicknesses of masseter and temporal muscles
The correlation between masseter and temporalis muscle thickness, considering patient posture and resting versus clenching states, was investigated.Interestingly, a very strong correlation (correlation coefficient r > 0.9) was found between muscle thickness during resting and clenching states.This suggests that in patients with TMD, thicker muscles observed during the resting period corresponded proportionally to thicker muscles during clenching.
In TMD_nonbruxers, during clenching in the upright position, a significantly strong correlation was found between the masseter muscle thickness and its resting state in the upright position (r = 0.699, p < 0.001).Similarly, a strong correlation was observed between the resting-state thickness of the temporalis muscle in the upright position and its thickness during clenching in the same position (r = 0.865, p < 0.001).Notably, in the supine position among TMD_non-bruxers, the resting-state thickness of the temporalis muscle exhibited the strongest positive correlation with its thickness during clenching in the same position (r = 0.922, p < 0.001).
In TMD_bruxers, during clenching in the upright position, a significantly strong correlation was observed between masseter muscle thickness and its resting state in the upright position (r = 0.824, p < 0.001).Similarly, a strong correlation was observed between the resting-state thickness of the temporalis muscle in the upright position and its thickness during clenching in the same position (r = 0.749, p < 0.001).Interestingly, in the supine position among TMD_bruxers, the resting-state thickness of the temporalis muscle showed the strongest positive correlation with its thickness during clenching in the same position (r = 0.933, p < 0.001) (Fig. 5).

Generalized linear model for masticatory muscle thickness changes
A generalized linear model was employed to investigate the factors influencing masseter and temporalis muscle thicknesses in the resting position while upright, aiming to quantify their respective effects.All previously examined clinical and ultrasonographic factors were used as explanatory variables, with masseter and temporalis Table 5. Muscle thickness differences between TMD subgroups based on DC/TMD.The results were obtained using t-tests.TMD, temporomandibular disorder; m, masseter muscle; statistical significance was set at p < 0.05.There was no significant difference in muscle thickness between the TMD subgroups (all p > 0.05).www.nature.com/scientificreports/muscle thicknesses as the outcome variables, enabling the creation of individual generalized linear models for each muscle.Concerning masseter muscle thickness, being female, in comparison to males, emerged as a predictive factor associated with reduced masseter muscle thickness (B = − 1.018, 95% CI − 1.855 to − 0.181, p = 0.017).Interestingly, bruxism significantly contributed to increased masseter muscle thickness (B = 0.868, 95% CI 0.567 to 1.169, p = 0.048).However, besides sex and bruxism, none of the clinical factors showed significant predictive power for masseter muscle thickness.
Furthermore, several clinical factors, including TMJ noise, TMD pain, tinnitus, sleep issues, occlusal dysesthesia, and TMD subgroups diagnosed using DC/TMD, did not exhibit significant explanatory power for the thickness of these two muscles (Table 6).

Discussion
We measured masticatory muscle thickness in both upright (seated in a unit chair) and supine positions to assess its potential variation due to body position.Comparative analyses evaluated muscle thickness differences between these positions.Additionally, we examined muscle thickness changes between resting and clenching states within each body position.We measured masseter and temporal muscle thickness in TMD_nonbruxer and TMD_bruxer groups, both at rest and during clenching in upright and supine positions.Overall, masticatory muscle thickness increased in TMD bruxers compared with TMD non-bruxers, predominantly during clenching rather than at resting.However, significant differences were found only in the masseter muscle, not the temporalis muscle.
The masseter muscle is of paramount importance during mastication, playing the most crucial role among the masticatory muscles.Electromyography studies revealed that the masseter muscle was more significantly affected than the temporalis muscle under maximal clenching or mastication 32,33 .Interestingly, in children, masseter muscle thickness had a positive correlation with bite force, while the anterior temporalis thickness was not associated with bite force 46 .The thickness of the masseter muscle in healthy individuals was significantly increased during contraction compared to at rest (from 1.09 to 1.40 cm), while the increase in the temporalis muscle was not statistically significant (from 0.88 to 0.98 cm) 34 .However, in adults aged 18-45 years, the differences in masseter and temporalis muscle thickness based on the presence or absence of bruxism during rest and dental clenching were not statistically significant 47 .In this study, notable differences in the masseter muscle were observed in TMD patients based on clenching and body posture.Mastication requires coordinated efforts and balance among various masticatory muscles.Given that the masseter muscle possesses larger muscle thickness, contributes more significantly to bite force, and plays a more crucial role in mastication compared to the temporalis muscle, along with anatomophysiological differences, conversely, the masseter muscle may be more susceptible to the influence of oral function/parafunction.
Bruxism defined as involuntary and excessive teeth grinding, involves forceful contact between upper (maxillary) and lower (mandibular) teeth surfaces.In 2013, an expert panel defined it as repetitive masticatory muscle activity 48 .This activity, termed bruxism, involves teeth clenching or grinding, accompanied by mandible straightening or pushing.It can occur during sleep (sleep grinding) or while awake (awake grinding) 12,48 .Bruxism involves involuntary and repetitive movements of the mandible muscles, with studies more frequently investigating its association with the masseter muscle than with the temporalis muscle among the main masticatory muscles 49 .Bruxism increases the risk of oral complications, including tooth wear, pain, and discomfort in the masticatory muscles and TMJ 50 .However, noting that not all cases of bruxism necessarily lead to these complications is important.When examining the EMG activity in the masseter and temporalis muscles, research revealed lower masseter EMG activity in patients with TMD than in healthy controls, whereas anterior temporalis EMG activity showed no difference between the two groups 51 .Palinkas et al. found no statistically significant differences in muscle thickness and EMG activity of the masseter and temporalis muscles based on the presence or absence of SB 47 .Further investigation is crucial to clarify whether the masseter muscle is more impacted than the temporalis muscle in patients with TMD or if the masseter muscle plays a more proactive role in bruxism than the temporalis muscle.
Sex-based differences in muscle thickness were evident solely in the masseter muscle, not the temporalis muscle.Among the four primary masticatory muscles, the masseter exerts the greatest force 6,52 .When comparing masseter and temporal muscle thickness between females and males based on body position and muscle status (resting or clenching), males displayed larger thickness in both muscles than females, with statistical significance observed only in the masseter muscle.Variations in skeletal muscle among sexes encompassed differences in muscle fiber count, Type I and II fiber distribution, although certain variances were specific to particular muscles 53 .An earlier EMG study reported higher masticatory activity in males than in females 51 .Specifically, in this study, masseter thickness during rest was measured at 12.46 ± 3.14 mm in males and 10.59 ± 2.83 mm in females.In a separate examination involving healthy Korean adults, ultrasonography recorded masseter muscle thickness at 11.3 ± 1.2 mm in males and 9.8 ± 1.3 mm in females 54 .These discrepancies likely stem from variations in age, TMD presence, measurement sites, and device types used..Stress is defined as an emotional experience wherein the perceived demands exceed the available resources.A systematic review and meta-analysis, involving three articles, found that individuals under stress were more prone to exhibiting bruxism compared with healthy individuals 21 .Moreover, somatic anxiety has been linked to SB 62 .However, there exists an alternate perspective suggesting that bruxism might act as an adaptive coping mechanism against psychological stress 59 .Although bruxism is not a specific disorder on its own 50 , its co-occurrence with TMD can exacerbate symptoms, underscoring the need for ongoing, comprehensive exploration of bruxism.Ultrasonography emerges as a promising diagnostic tool for TMD, offering advantages over MRI, the current gold standard for TMJ assessment.Unlike CBCT, optimized for bone structures, ultrasonography is cost-effective and widely available in outpatient clinics 63 .The test is non-invasive, quick (typically 10-15 min), and welltolerated even by diverse age groups.The protocol for ultrasonography entails no radiation or associated risks, while being painless.It allows dynamic real-time assessment with the mouth open or closed and facilitates direct communication with the patient to focus on painful areas.Importantly, sedation is not necessary for children or older adults 64 .Furthermore, Ariji et al. compared ultrasound examination and MRI in patients, highlighting their potential for diagnosing or treating masticatory muscle pain 65 .Muscle thickness investigation has traditionally been straightforward [66][67][68] .Given the advantages of ultrasonography and its nascent stage of implementation in TMD, more reliable studies focusing on the anatomical and pathological traits of the TMJ and masticatory muscle are required.Although this study demonstrated high agreement among examiners in measuring muscle thickness, its ability to identify active inflammation and arthritic changes as accurately as MRI or CBCT remains uncertain.Furthermore, developing and establishing protocols for easier and quicker detection of TMJ effusion, arthritis, and myalgia in patients with TMD is necessary.
This study has several limitations.First, bruxism was not diagnosed using polysomnography but relied on patient self-reports and a questionnaire.Despite numerous prior studies relying on self-reported bruxism [69][70][71] , this approach may pose a significant limitation in affirming the robustness of the research.Furthermore, muscle thickness was not investigated by segregating patients with TMD based on signs and symptoms on affected versus non-affected sides.Instead, for research purposes, we averaged the right and left muscle thickness.Consequently, comparisons between muscle values from the TMD patient group and healthy controls were not feasible.Among masticatory muscles, the lateral and medial pterygoid muscles have limited accessibility to ultrasound probes.Nevertheless, considering the involvement of the superior head of the lateral pterygoid muscle in clenching and the role of the medial pterygoid muscle in mandibular elevation and mastication [72][73][74][75] , further research on these muscles is also warranted.Nevertheless, this study is pioneering in directly comparing masseter and temporalis muscle thickness in TMD_bruxers and TMD_nonbruxers, considering patient posture and clenching condition.

Conclusions
In this study, we aimed to assess ultrasonography-based changes in the thickness of major masticatory muscles-masseter and temporalis-in various body positions and during occlusal resting and clenching.Clenching led to increased masseter muscle thickness compared with that at resting, with bruxism further contributing to this increase.However, the temporalis muscle thickness showed no significant change owing to these factors.Posture variations only affected the masseter muscle thickness, not the temporalis.In patients with TMD, bruxism showed a positive correlation with tinnitus, muscle stiffness, sleep issues, psychological stress, and limited mouth opening.To enhance understanding, longitudinal studies tracking masticatory muscle thickness changes over time, considering clinical factors, are warranted, preferably with a multicenter approach.

Figure 1 .
Figure 1.Ultrasound image paired with a schematic diagram of ultrasound observation of the main masticatory muscles-masseter and temporalis muscles.Thickness measurement of (A) masseter muscle in the upright position, (B) temporal muscle in the upright position, (C) masseter muscle in the supine position, and (D) temporal muscle in the supine position.Muscle thickness was measured sequentially in both resting and clenching conditions of the face and teeth for each position.

Table 2 .
Distribution of chief complaints and contributing factors for TMD.The results were obtained using a two-sided chi-square analysis.TMD, temporomandibular disorder; statistical significance was set at p < 0.05.*p < 0.05, **p < 0.01.The significant values are indicated in bold font.

Table 3 .
Masseter and temporal muscle thickness during resting and clenching in patients with TMD.The results were obtained using t-tests.TMD, temporomandibular disorder; m, masseter muscle.a Difference between muscle thickness at clenching and that at rest.Statistical significance was set at p < 0.05.*p < 0.05, **p < 0.01, ***p < 0.001.The significant values are indicated in bold font.

Figure 3 .
Figure 3. Differences in muscle thickness between bruxers and non-bruxers among patients with TMD.

Figure 4 .
Figure 4. Differences in muscle thickness between sexes in patients with TMD.

Figure 5 .
Figure 5. Correlation of thicknesses in each condition of masseter and temporal muscles.The results were obtained using Spearman's correlation analysis; U, upright position; S, supine position; R, resting; C, clenching; m, muscle.Statistical significance was set at p < 0.05.Significant values are indicated in bold.

Table 1 .
Demographics and TMD diagnosis based on DC/TMD among bruxers and nonbruxers.a Results were obtained using t-tests.b Results were obtained using a two-sided chi-square analysis.TMD, temporomandibular disorder; VAS, visual analog scale; Chronicity, chronic state that has elapsed more than 6 months from the onset of TMD symptoms; DC/TMD, diagnostic criteria for TMD.Statistical significance was set at p < 0.05.*p < 0.05.**p < 0.01.The significant values are indicated in bold font.